US20030171290A1 - Peptides presented by cells - Google Patents

Peptides presented by cells Download PDF

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US20030171290A1
US20030171290A1 US10/343,443 US34344303A US2003171290A1 US 20030171290 A1 US20030171290 A1 US 20030171290A1 US 34344303 A US34344303 A US 34344303A US 2003171290 A1 US2003171290 A1 US 2003171290A1
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peptides
cells
protein
polypeptide
mhc
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Francis Carr
Graham Carter
Koen Hellendoorn
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Merck Patent GmbH
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Merck Patent GmbH
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

Definitions

  • the present invention relates to methods to determine peptides presented on the surface of mammalian cells following addition to the cells of a protein.
  • the present invention also relates to diagnostic tests based on the determination of such peptides or modified molecules resulting from determination of such peptides, such as pharmaceutical entities preferably having specific biological activity and reduced or enhanced immunogenicity when compared with the corresponding non-modified molecules.
  • the methods according to this invention are preferably established with tools using mass spectroscopy (MS).
  • proteins are subsequently degraded and, commonly, peptide fragments of such proteins appear at the cell surface, often associated with other proteins.
  • peptide fragments of a protein can be degraded and certain peptides subsequently become associated with major histocompatability complex (MHC) molecules which, in many cases, can be recognised by T cells in order to initiate an immunological response.
  • MHC major histocompatability complex
  • Such an immunological response can be beneficial, for example where the immune system counteracts cells producing the specific protein from where the peptide fragments are derived, or can be detrimental, for example where the immune system produces antibodies which bind to the specific protein and limit its activity (such as with a pharmaceutical protein).
  • a vaccine molecule For a vaccine molecule, it is a particular object to identify peptides presented by MHC molecules following uptake or production of a protein by cells and to use one or more of such peptides within a vaccine molecule in order to stimulate the immune system.
  • a diagnostic test it is a particular object to determine peptides presented by MHC molecules following uptake or production of a protein by cells and to use such determination as the basis for a test for the detection of a biological or physiological event, for example for the detection of an infection.
  • the present invention provides for the accurate and comprehensive analysis of peptides on the surface of cells and in particular peptides in association MHC molecules. Determination of the profile or identity of peptides on the cell surface will be especially performed mass spectrometry (MS).
  • MS mass spectrometry
  • Salter et al provide genetically engineered temperature-sensitive MHC class I molecules enabling the facile removal of peptides bound to the mutant MHC class I molecule from engineered cells grown in vitro.
  • 5,734,023 and others provide methods for the purification of particular MHC peptide complexes for use as therapeutic entities for example in the treatment of auto-immune diseases and, Deshpande et al [WO9740852] disclose modified peptides with enhanced binding to an MHC class II protein and where the whole complex is provided as a recombinant fusion protein also intended for use in the treatment of diseases associated with auto-reactive T cells.
  • WO9734143 and U.S. Pat. No. 5,792,604 provide methods for identifying MHC class I restricted antigens endogenously processed by cellular secretory pathway.
  • This biological assay requires primed cytotoxic T cells and a source of “indicator” target cells onto which lysis is directed by the presence of processed peptides secreted into the medium.
  • the scheme has utility in the identification of substances able to influence the endogenous processing pathways of the donor cells.
  • Other complex biologically based schemes for the identification MHC class I and MHC class II restricted T-cell epitopes include WO00/67761 where is provided a method exploiting genetic vaccination and isolation of dendritic cells and splenocytes for conducting biological T-cell activation assay in vitro.
  • MS-based instruments may be applied to the analysis of whole cells or cell extracts and the data incorporated into a pathway for the rational development of therapeutic molecules or diagnostic assays.
  • ESI electrospray ionisation
  • MALDI matrix assisted laser de-sorption ionisation
  • ESI is a continuous flow technique and as such is readily connected to up-stream sample separation technologies such as liquid chromatography (LC) or capillary electrophoresis (CE) [Cao & Moini (1998) Am. Soc. Mass Specrom. 9: 1081-1088].
  • LC liquid chromatography
  • CE capillary electrophoresis
  • mass analysis is most usually conducted by a time of flight (TOF) instrument where the flight time from the ionising laser pulse to detection is measured enabling calculation of the mass to charge ratio (m/z).
  • TOF time of flight
  • Some models enable analysis of post-source decay ion fragments for peptide sequence derivation and other structural determinations.
  • the continuous flow nature of ESI based instruments result in the use of scanning analysers (e.g. quadrupole or magnetic sector mass analysers) although other instrument formats may be exploited.
  • a typical format is the tandem MS system whereby dual mass analysers are arranged in tandem and interconnected via a “collision cell” containing molecules of inert gas. The latter feature enables generation of collision induced decay ions and the determination of peptide sequence from the given input sample.
  • Multiple and hybrid instrumental formats may be arranged and exploited for the analysis of cell surface (MHC) associated peptides.
  • MHC cell surface
  • Methods 246: 1-12] exploit MALDI-TOF in the analysis of self peptides acid eluted from MHC class II allele DRB1*0401 following treatment of the cells with measles virus vaccine.
  • Sickman et al [Sickman, A. et al (2000) Analyst 125: 569-573] describe the identification of MHC class II bound peptides from rat Langerhans cells using a combination of LC and MALDI-MS techniques.
  • the present invention however, incorporates and extends all such prior art by for the first time providing a generalised scheme for the elucidation of peptide species binding to MHC class I and MHC class II molecules using MS based instrumentation and in the first embodiment provides methods for the removal of the binding interaction by amino acid substitution within the peptide sequence for the purpose of developing a therapeutic protein with a reduced or absent ability to provide an MHC-mediated immune response on administration to a subject, most preferably a human subject. It is an objective of the present invention to provide a method for the development of a therapeutic molecule with reduced capacity to elicit an immune response upon administration to a subject.
  • the exogenous protein may be a purified preparation extracted from a mammalian source such as a cell line or tissue ex vivo, or the protein may be a recombinant preparation purified from any biological source engineered to express said protein.
  • the protein may be representative of a naturally occurring protein or a fusion of one or more naturally occurring proteins.
  • the protein may be in vitro derived being an assemblage of naturally occurring elements or wholly synthetic or designed and have no counterpart in nature.
  • the protein may be exemplary of a class of proteins such as antibody molecules and their derivatives, cytokines, growth factors, leukotrines, haemostatic factors or may be a cell toxin or have enzymatic capacity or function. Most preferably the protein is a therapeutic protein.
  • T cell epitopes A principal factor in the induction of an immune response is the presence within the protein of peptides that can stimulate the activity of T cell via presentation on MHC class II molecules, so-called T cell epitopes.
  • T cell epitopes In order to eliminate or reduce immunogenicity, it is desirable to identify and remove T cell epitopes from the protein. It is an object of the present invention to provide for methods to enable the detection and identification of T cell epitopes.
  • MHC Class II molecules are expressed by professional antigen presenting cells (APCs), such as macrophages and dendritic cells amongst others.
  • APCs professional antigen presenting cells
  • the ability of a peptide to bind a given MHC class II molecule for presentation on the surface of an APC is dependent on a number of factors most notably its primary sequence. This will influence both its propensity for proteolytic cleavage and also its affinity for binding within the peptide binding cleft of the MHC class II molecule.
  • the MHC class II/peptide complex on the APC surface presents a binding face to a particular T cell receptor (TCR) able to recognise determinants provided both by exposed residues of the peptide and the MHC class II molecule.
  • TCR T cell receptor
  • computational methods for predicting potential T-cell epitopes or recognising sequence motifs in experimentally determined T-cell epitopes are computational methods for predicting potential T-cell epitopes or recognising sequence motifs in experimentally determined T-cell epitopes.
  • Schemes include techniques to predict MHC class II-binding peptides as provided in WO98/52976 where a computational threading approach identifies peptide sequences with the potential to bind only a sub-set of possible human MHC class II DR allotypes.
  • T cell epitope identification is the first step to epitope elimination as recognised in WO98/52976 and WO00/34317.
  • predicted T cell epitopes are removed by the use of judicious amino acid substitution within the primary sequence of the therapeutic protein. It is an objective of the present invention to provide methods for the development of modified proteins in which the immune characteristic is modified by means of reduced numbers of potential T-cell epitopes.
  • the identified sequences being present on the surface of MHC bearing cells or bound to MHC preparations following the natural intracellular processing events present within any competent APC or similar cell.
  • peptides identified under the scheme of the present invention can be detected from any MHC allotype or a mixture of allotypes including for MHC class II, those of the DR, DQ or DP specificities.
  • the present invention enables the detection of peptides displayed in the surface of cells and in particular in association with MHC molecules of the class I and class II designations, it is not intended to be limited to whole cells but also includes the analysis of peptides on the surface of exosomes.
  • Peptide-MHC complexes are present in high concentration on the surface of exosomal vesicles derived from cells normally expressing MHC class II molecules. Under certain circumstances the output of exosomes may be increased from the surface of an APC and there are recognised procedures for the enrichment or isolation of exosomes [Raposo, G. et al (1996) J. Exp. Med. 183: 1161-1172; Clayton, A. et al (2001) J. Immunol. Methods 247: 163-174]. Analysis of APC preparations and preparations of exosomal particles derived from APC equally therefore fall under the scope of the present invention.
  • a corresponding method further comprising the following steps:
  • a corresponding method, wherein the analysis of said cells or exosomal vehicles thereof of step (ii) is performed by using mass spectroscopy (MS), preferably MALDI-MS and ESI-MS.
  • MS mass spectroscopy
  • step (i) A corresponding method, wherein the peptides bound on the surface of the cells or exosomal vesicles according to step (i) are in association with MHC molecules and wherein the analysis of said cells or exosomal vesicles thereof according to step (ii) is performed by using MS.
  • step (i) A corresponding method, wherein the peptides bound on the surface of the cells or exosomal vesicles according to step (i) are products of an intracellular peptidase and trafficking pathway.
  • a corresponding method for the development of a pharmaceutical protein or polypeptide having a reduced immunogenicity wherein the modification of the immunogenic peptides is performed by eliminating or reducing their binding to MHC molecules, optionally by testing the modified peptides for binding to the cell surface as indicated in claim 1.
  • a corresponding method, wherein the elimination or reduction of the binding of the peptides to MHC molecules is performed by substituting, inserting or deleting one or more amino acid residues within the sequence region of the peptide within the pharmaceutical protein or polypeptide.
  • a method for the development of a pharmaceutical protein or polypeptide having an enhanced immunogenicity wherein the modification of the peptides are performed by enhancing their binding to MHC molecules, optionally by testing the modified peptides for binding to the cell surface as indicated in claim 1.
  • a corresponding method wherein the enhancement of the binding of the peptides to MHC molecules is performed by substituting, inserting or deleting one ore more amino acid residues within the sequence region of the peptide increasing the activity of the peptide to act as a T-cell epitope, and/or broadening the range of MHC types for which the T-cell epitope is restricted, and/or combining several otherwise disparate epitopes into a single entity.
  • a corresponding method, wherein the MHC molecules are HLA-DR, HLA-DQ and HLA-DP.
  • (ii) either, (a) producing a profile of peptides which appear on the cell surface and, optionally, comparing with other profiles to identify an abnormality or disease associated with the human cells, or, (b) determining the sequence of specific peptides on the cell surface as a means to determining specific peptides which might used to identify an abnormality or disease associated with the human cells.
  • a population of mammalian cells is incubated with a test protein or transfected with a gene encoding a test protein and, following uptake or expression of the protein and its degradation, the profile or identity of peptides on the cell surface is determined.
  • MHC molecules especially multiple MHC molecules, will present such peptides.
  • Such cell populations can either be obtained by sampling multiple cell populations from the world population or by using cells that have been engineered to produce multiple MHC types. Of particular use are cell lines where no endogenous MHC molecules are produced and which display a low background of peptides on the cell surface.
  • a particular application is to generate a protein whereby epitopes for activation and/or stimulation of helper T cells are removed.
  • the primary interest is peptides presented by MHC class II.
  • MHC class II there is a need for more accurate and comprehensive methods than currently available for predicting or detecting peptides which bind to one or more of the numerous MHC class II allotypes and genotypes in order to comprehensively identify potential T cell epitopes.
  • Major benefits of the availability of such an approach is in the generation of pharmaceuticals with all major (or all) T cell epitopes identified and then removed.
  • a preferred method for the development of a pharmaceutical using the present invention comprises the following steps:
  • test protein for potential use as a pharmaceutical, add the protein to a selection of human cell lines or human cell samples
  • a vaccine molecule or molecule able to function as a vaccine may be developed.
  • the vaccine is most preferable for use in humans although the scheme may equally be applied to the development of vaccine molecules for the treatment or prevention of disease in non-human species.
  • a particular application is to generate a peptide or protein whereby major epitopes for activation and/or stimulation of T cells are present, primarily epitopes for helper T cells but also, for applications requiring cell killing, epitopes for cytotoxic T cells (primarily MHC class I restricted).
  • peptides within the protein that can be presented by one or more of the numerous MHC allotypes and genotypes and to then select from these peptides one or more epitopes for inclusion in the final vaccine molecule.
  • epitopes can comprise peptides restricted by MHC class I or MHC class II or both.
  • the present invention provides for such identification of peptides presented by MHC class I and class II and is thus the basis for development of more effective vaccines.
  • MHC class I and MHC class II epitopes are the principal origin of the protein from which the peptide in the MHC-peptide complex is usually derived. It is recognised that exogenous proteins give rise predominantly to peptides in association with MHC class II, whereas endogenous proteins expressed from within a cell are processed according to a different peptidase and intracellular trafficking pathway to result predominantly in association with MHC class I molecules on the cell surface. For either exogenous or endogenous proteins, during the sequence of processing steps, certain peptide sequences from the protein that might normally bind to MHC may be destroyed so that certain T cell epitopes are not produced in vivo.
  • peptides identified from the cell surface are those that have been selected by the APC (or other cell) as capable of passage via the intracellular processing and trafficking pathways. As the invention is focussed to the identification of the real products of the processing events, the invention therefore reduces the rate of false positive and false negative epitopes identified using predictive or in vitro methods of epitope identification.
  • a preferred method for the development of a vaccine using the present invention therefore comprises the following steps:
  • the analysis of cell surface peptides will also include the optional step of enriching MHC molecules, for example using immunoaffinity columns, prior to peptide analysis.
  • the method of the invention for analysis of cell surface peptides has the particular advantage of providing for analysis of binding to allotypes other than DR. Analysis of peptide presentation by such allotypes as DQ and DP has been very difficult as there are no predictive method available especially for DQ where both chains of the MHC molecule are thought to contribute to binding (in contrast to just the ⁇ chain in DR).
  • a particular application of this embodiment is to generate a profile of peptides presented on the cell surface in order to identify the abnormal presence, absence or pattern of peptides that might indicate a particular disease or cellular insult.
  • the presence of a “diagnostic peptide” or “indicator peptide” may be made in isolation by the registration of the indicator peptide mass in the mass spectrum of an MS instrument, or by record of the indicator peptide sequence in a CID (or similar) spectrum.
  • the presence of the indicator peptide may be indicated with reference to a mass spectrum derived from a reference sample not contacted with the exogenous protein or agent.
  • a comparative analysis is of particular value in instances where the mass(es) of the indicator peptides are not known or predicted-by any means. Comparative analysis may be conducted in silico by subtraction of identical mass peaks and would be or particular value where the loss of a particular mass peak is the diagnostic indicator. Others too have exploited MS techniques in comparative analysis of biological samples. However, in the case of Liotta et al [WO0049410], such comparative analysis is focussed to the protein content of individual cells obtained by laser capture micro dissection. This is distinct from the current invention, where the diagnosis (comparative, subtractive or otherwise) is according to the peptides detected from the surface and particularly peptides in association with MHC molecules on the cell surface.
  • a preferred method for the development of a diagnostic test comprises the following steps:
  • [0116] analyse appropriate human cells for surface peptides particularly using MALDI-MS directly on cells or, alternatively, using MALDI-MS on cell fractions especially MHC fractions, or alternatively, eluting peptides from the cell surface prior to analysis particularly by MALDI-MS or ESI-MS.
  • [0117] either, produce a profile of peptides which appear on the cell surface and, if necessary, compare with other profiles to identify an abnormality or disease associated with the human cells, or, determine sequence of specific peptides on the cell surface as a means to determining specific peptides which might used to identify an abnormality or disease associated with the human cells.
  • EBV transformed human B cell lines JESTHOM and BSM were obtained from ECACC (Porton Down, UK).
  • B cell line JESTHOM is homozygous for HLA-DR1*0101 and line BSM is homozygous for DRB1*0401.
  • Cell lines were cultured in vitro using conditions recommended by the supplier.
  • Mouse NSO cells (ECACC #85110503) cultured using the suppliers recommended conditions were used as a negative control cell line.
  • Synthetic peptides were obtained from Zeneca (Zeneca LSM, Northwich, UK).
  • Peptide HA1 was a 13mer corresponding to residues 307-319 of influenza virus hemagglutinin protein (Rothbard, J. B. et al (1988) Cell 52: 515).
  • Peptide MP1 was 13mer corresponding to residues 17-29 of influenza virus matrix protein [Rothbard, J. B. et al (1988) ibid]. Whilst both peptides are known to bind a number of different HLA-DR specificities, differential binding is seen with the DR4 allotype. MP1 binds to DR4 whereas MP1 shows no significant binding to the DR4 specificity [Busch R. et al (1990) Int. Immunol. 2: 443].
  • the antibody was purified from hybridoma LB3.1 conditioned medium using protein A sepharose (Millipore, Conset, UK) affinity chromatography and procedures recommended by the supplier. The hybridoma had been maintained using standard conditions. LB3.1 was co-incubated with the peptide and cells at a maximum concentration of 10 ⁇ g/ml.
  • Each sample was subsequently mixed vigorously for 5 s before sample spotting to the MALDI instrument sample plate.
  • the two-layer method of sample spotting was used, where 1 ⁇ l acid matrix solution [0.1M sinapinic acid in a 1:1:1 mixture of acetonitrile, methanol and water] was dried onto the MS sample plate. This was followed by 1 ⁇ l of the cells and a further 1 ⁇ l of sinapinic acid matrix solution.
  • a Voyager DE mass spectrometer (Perseptive Biosystems, Foster City, Calif., USA) was used in positive ion mode. The instrument was calibrated with a mixture of horse cardiac apomyglobin and bovine serum albumin (Sigma, Poole, UK) and checked less than 30 minutes before each analysis to be within 0.1% mass accuracy for each standard. The sample spots were air dried and analysed in positive ion mode. The delayed extraction was set to 150 ns at 25 kV. The low mass gate was set to 600 Da. A total of 125 laser shots were accumulated from each sample.
  • HLA-DR1*0101 was purified from JESTHOM cell membranes by immunoaffinity chromatography.
  • HLA DRB1*0401 was purified from BSM cells. Solubilised cell membranes were prepared and processed as previously described [Gorga et al (1987) J. Biol. Chem. 262: 16087-16094; Sette et al (1989) J. Immunol. 42: 35-40].
  • the anti-DR monoclonal antibody LB3.1 [ATCC number HB-298] was used as the immunoaffinity reagent.
  • the antibody was purified from hybridoma LB3.1 conditioned medium using protein A sepharose (Millipore, Conset, UK) affinity chromatography and procedures recommended by the supplier.
  • LB3.1 antibody was coupled to sepharose 4B (Pharmacia Biotech, St. Albans, UK) using the Linx system provided by InVitrogen (InVitrogen, Groningen NL) and conditions recommended by the supplier.
  • Synthetic peptides HA1 and MP1 as described in example 1 were incubated with a 50 ⁇ l aliquot of the HLA-DR1*0101 preparation. Peptides were incubated in peptide binding buffer (50 mM Phosphate citrate buffer at pH 4.0; 0.1% NP40) at a final concentration of 50 ⁇ M for 26 hours at 37° C. Unbound peptide was removed by ultrafiltration using microcon centrifugal filtration cells (Millipore, USA) and multiple cycles (minimum of four) of washing with PBS. The final volume was reduced to 20 ⁇ l. In some experiments peptides were eluted from the MHC before MALDI-tof analysis.
  • peptide binding buffer 50 mM Phosphate citrate buffer at pH 4.0; 0.1% NP40
  • Human dendritic cells were enriched from 40 ml peripheral blood samples obtained from healthy donors. The blood was anticoagulated using heparin and a mononuclear cell fraction prepared using histopaque 1077 (Sigma, Poole, UK) density gradient medium and conditions recommended by the supplier. Dendritic cells were obtained by negative selection using an immunomagnetic separation procedure with all reagents and conditions provided by Mitenyi Biotec (Bisely, UK). The dendritic cells were eluted into multiwell tissue culture dishes for treatment with protein antigen. Cells were maintained in RPMI medium supplemented with 10% (v/v) foetal bovine serum and standard antibiotics during the course of the experiments. All reagents were from Life Technologies (Paisley, UK).
  • a preparation of recombinant staphylokinase was used in these studies.
  • the wild-type staphylokinase gene was synthesised under contract by Genosys Biotechnologies Ltd (Cambridge, UK).
  • the gene was constructed by polymerase chain reaction using overlapping synthetic primers and the sequence as given by Collen et al [Collen D. (1996) Circulation 94: 197-206].
  • the synthetic gene was cloned as a 453 bp EcoRI-HinDIII restriction fragment into bacterial expression vector pMEX (MoBiTec, Gottingen, Germany).
  • the pMEX/staphylokinase gene was transformed into competent E.coli strain TG1 by standard techniques and a single transformed clone secreting active staphylokinase was selected using a fibrin plate assay [Astrup, T. et al (1952) Arch. Biochem. Biophys. 40: 346-351; Collen, D. et al (1992) Fibrinolysis 6: 203-213].
  • the best expressing clone was grown up and recombinant protein was purified from the culture supernatant using sequential column chromatography and methods described previously [Collen, D. et al (1992) ibid; Schlott, B. et al (1994) Biotechnology 12: 185-189].
  • Dendritic cells were incubated with purified staphylokinase at a concentration of 100 ⁇ g/ml and incubation conducted overnight. In some experiments, antigen processing was blocked using inhibitor cocktails added to the culture medium. Inhibitors were added 1 hour before the addition of the test protein at the following concentrations: ammonium chloride 50 mM; sodium azide 1 mg/ml; chloroquine and colchicine 500 ⁇ M. All compounds were from Sigma (Sigma, Poole, UK).
  • Ion peaks attributed to staphylokinase were identified in spectra obtained from cells treated with staphylokinase and not seen in the spectra of cells treated with metabolic inhibitors or control cells not treated with staphylokinase.
  • the column was eluted using a standard elution gradient ranging from a solvent solution of 95% H 2 O, 5% acetonitrile, 0.1% formic acid to a solvent solution of 5% H 2 O, 95% acetonitrile, 0.1% formic acid.
  • Electrospray MS and MS/MS data were acquired on a Micromass Q-T of 2 instrument (Micromass, Wythenshawe, UK) fitted with a Z-spray nanoflow electrosparay ion source.
  • the instument was operated in the positive ion mode with a source temperature of 80° C., a counter gas flow rate of 40 L/hour and with a potential of 2800V applied to the nanospray continuous LC probe. All data were acquired with the instrument operating in automatic data dependent switching mode.
  • the instrument was calibrated with a two point calibration selected fragment ions that resulted from the collision-induced decomposition of glufibrinopeptide b. All data was processed automatically by means of ProteinLynx software, protein identification was achieved by analysis with the ProteinLynx Global Server search algorithm (Micromass, Wythenshawe, UK).
  • Spectra were collected and analysed for the presence peptide masses attributable to the test protein. These were identified by comparison to spectra obtained from control cells including cells in which antigen processing was blocked. Following treatment with staphylokinase peptide masses attributed staphylokinase were identified. Equivalent ion peaks were not evident in cells not treated with staphylokinase or with cells treated with metabolic inhibitors.

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US20040235062A1 (en) * 2001-06-19 2004-11-25 Terumi Nakajima Method of analyzing protein occurring in cell or substance interacting with the protein
US20090061478A1 (en) * 2006-01-30 2009-03-05 Lene Have Poulsen High-Speed Quantification of Antigen Specific T-Cells in Whole Blood by Flow Cytometry
US20100248257A1 (en) * 2007-07-03 2010-09-30 Dako Denmark A/S Compiled Methods for Analysing and Sorting Samples
US20110195435A1 (en) * 2008-09-20 2011-08-11 Andrew Kelvin Sewell Use of a protein kinase inhibitor to detect immune cells, such as t cells
US20110212090A1 (en) * 2008-07-23 2011-09-01 Dako Denmark A/S Combinatorial Analysis and Repair
US8268964B2 (en) 2007-03-26 2012-09-18 Dako Denmark A/S MHC peptide complexes and uses thereof in infectious diseases
US20170199961A1 (en) * 2015-12-16 2017-07-13 Gritstone Oncology, Inc. Neoantigen Identification, Manufacture, and Use
CN108473973A (zh) * 2016-09-30 2018-08-31 赛尔莱克斯生命科学公司 包含负载蛋白的外泌体的组合物以及制备和递送该组合物的方法
US10369204B2 (en) 2008-10-02 2019-08-06 Dako Denmark A/S Molecular vaccines for infectious disease
US10611818B2 (en) 2007-09-27 2020-04-07 Agilent Technologies, Inc. MHC multimers in tuberculosis diagnostics, vaccine and therapeutics
US10968269B1 (en) 2008-02-28 2021-04-06 Agilent Technologies, Inc. MHC multimers in borrelia diagnostics and disease
US11264117B2 (en) 2017-10-10 2022-03-01 Gritstone Bio, Inc. Neoantigen identification using hotspots
US11885815B2 (en) 2017-11-22 2024-01-30 Gritstone Bio, Inc. Reducing junction epitope presentation for neoantigens
US11992518B2 (en) 2008-10-02 2024-05-28 Agilent Technologies, Inc. Molecular vaccines for infectious disease
US12258373B2 (en) 2018-12-17 2025-03-25 Immudex Aps Panel comprising Borrelia MHC multimers

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AU2004281634B2 (en) * 2003-09-03 2011-01-27 Dendritherapeutics, Inc. Multiplex vaccines
DE602004030811D1 (de) 2003-10-16 2011-02-10 Micromet Ag Multispezifische deimmunisierte cd3-bindende moleküle
JP5822841B2 (ja) 2009-11-05 2015-11-24 テバ・ファーマシューティカルズ・オーストラリア・ピーティワイ・リミテッド 変異型のｋｒａｓまたはｂｒａｆ遺伝子を含むがんの治療
US20150017136A1 (en) * 2013-07-15 2015-01-15 Cellectis Methods for engineering allogeneic and highly active t cell for immunotherapy
MX2015015638A (es) * 2013-05-13 2016-10-28 Cellectis Metodos para diseñar celulas t altamente activas para inmunoterapia.

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040235062A1 (en) * 2001-06-19 2004-11-25 Terumi Nakajima Method of analyzing protein occurring in cell or substance interacting with the protein
US20090061478A1 (en) * 2006-01-30 2009-03-05 Lene Have Poulsen High-Speed Quantification of Antigen Specific T-Cells in Whole Blood by Flow Cytometry
US10336808B2 (en) 2007-03-26 2019-07-02 Dako Denmark A/S MHC peptide complexes and uses thereof in infectious diseases
US8268964B2 (en) 2007-03-26 2012-09-18 Dako Denmark A/S MHC peptide complexes and uses thereof in infectious diseases
US10030065B2 (en) 2007-07-03 2018-07-24 Dako Denmark A/S MHC multimers, methods for their generation, labeling and use
US20100248257A1 (en) * 2007-07-03 2010-09-30 Dako Denmark A/S Compiled Methods for Analysing and Sorting Samples
US10611818B2 (en) 2007-09-27 2020-04-07 Agilent Technologies, Inc. MHC multimers in tuberculosis diagnostics, vaccine and therapeutics
US10968269B1 (en) 2008-02-28 2021-04-06 Agilent Technologies, Inc. MHC multimers in borrelia diagnostics and disease
US20110212090A1 (en) * 2008-07-23 2011-09-01 Dako Denmark A/S Combinatorial Analysis and Repair
US10722562B2 (en) 2008-07-23 2020-07-28 Immudex Aps Combinatorial analysis and repair
US9404916B2 (en) 2008-09-20 2016-08-02 University College Cardiff Consultants Limited Use of a protein kinase inhibitor to detect immune cells, such as T cells
US20110195435A1 (en) * 2008-09-20 2011-08-11 Andrew Kelvin Sewell Use of a protein kinase inhibitor to detect immune cells, such as t cells
US11992518B2 (en) 2008-10-02 2024-05-28 Agilent Technologies, Inc. Molecular vaccines for infectious disease
US10369204B2 (en) 2008-10-02 2019-08-06 Dako Denmark A/S Molecular vaccines for infectious disease
US20170199961A1 (en) * 2015-12-16 2017-07-13 Gritstone Oncology, Inc. Neoantigen Identification, Manufacture, and Use
US10847252B2 (en) 2015-12-16 2020-11-24 Gritstone Oncology, Inc. Neoantigen identification, manufacture, and use
US10847253B2 (en) 2015-12-16 2020-11-24 Gritstone Oncology, Inc. Neoantigen identification, manufacture, and use
US11183286B2 (en) 2015-12-16 2021-11-23 Gritstone Bio, Inc. Neoantigen identification, manufacture, and use
CN108473973A (zh) * 2016-09-30 2018-08-31 赛尔莱克斯生命科学公司 包含负载蛋白的外泌体的组合物以及制备和递送该组合物的方法
US11264117B2 (en) 2017-10-10 2022-03-01 Gritstone Bio, Inc. Neoantigen identification using hotspots
US11885815B2 (en) 2017-11-22 2024-01-30 Gritstone Bio, Inc. Reducing junction epitope presentation for neoantigens
US12258373B2 (en) 2018-12-17 2025-03-25 Immudex Aps Panel comprising Borrelia MHC multimers

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DE60114018T2 (de) 2006-06-29
MXPA03000901A (es) 2003-06-24
NO20030495L (no) 2003-01-31
PL358874A1 (en) 2004-08-23
CZ2003464A3 (cs) 2003-06-18
DE60114018D1 (de) 2006-02-23
ATE306498T1 (de) 2005-10-15
HUP0301581A2 (hu) 2003-08-28
NO20030495D0 (no) 2003-01-31
SK1982003A3 (en) 2003-08-05
KR20030037272A (ko) 2003-05-12
CA2417767A1 (en) 2002-02-14
BR0112925A (pt) 2003-08-26
ZA200301637B (en) 2004-02-03
AU2001283958A1 (en) 2002-02-18
GB0018901D0 (en) 2000-09-20
WO2002012899A2 (en) 2002-02-14
CN1511164A (zh) 2004-07-07
RU2003105806A (ru) 2004-07-20
JP2004506202A (ja) 2004-02-26
EP1305343A2 (de) 2003-05-02
EP1305343B1 (de) 2005-10-12
WO2002012899A3 (en) 2002-11-07

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